**4. Conclusions**

118 New Approaches to the Study of Marine Mammals

the cell cultures are environmentally relevant concentrations.

The next challenge is to compare the tissue culture doses to the levels in the whales. Comparisons can be made to gain toxicological context; however, one must always bear in mind that cells grown on a dish are in a different environmental context than cells in a tissue and so the comparisons will not be precise. To contextualize the chromium toxicity data, we have converted our treatment concentrations to parts per million and converted the levels observed in the biopsies to molarity. This way we can determine if the levels we are using in

Our sodium chromate treatments convert to a range of 0.052-1.3 ppm and our lead chromate treatments convert to a range of 0.34-6.8 ppm (Table 1). Considering that average tissue levels for sperm whales and right whales were 9.3 and 7.0 ppm, respectively, our treatment concentrations are well below the average levels measured in whale skin (Tables 1 and 2). In fact, if we were to treat the cell cultures with the lowest sperm whale regional average measured in Sri Lankan waters (63.8 uM) we would kill all of the cells because our highest treatment concentration of 25 uM was highly cytotoxic (Figure 2) as measured by our cytotoxicity assay. Even our lowest detectable level in the sperm whale, 0.9 ug/g, is equivalent to treating cells with 6.38 uM and would induce cytotoxicity in approximately 65% of North Atlantic right whale cells and 90% of sperm whale cells. This level of treatment would also induce DNA damage in approximately 20% of both sperm whale and right whale metaphases. Given that our experimental doses are so much lower than the levels found in whales; this outcome raises concern about the impact of chromium pollution on whales regardless of the

difference in environmental context between cells in a dish and cells in a tissue.

**Lead Chromate (ug/cm2)** 

**Table 1.** Chromate Treatment Conversions.

**Sodium Chromate (uM) Total Chromium(ppm)** 1 0.05 2 0.10 2.5 0.13 3 0.16 5 0.26 10 0.52 25 1.3

> 0.5 0.34 1 0.68 2 1.4 3 2.0 4 2.7 5 3.4 10 6.8

**Total Chromium (ppm)** 

**3.3. Toxic context** 

Ocean pollution is emerging as a global priority. No longer can the world's oceans be considered an easy collective dumping site because even the most remote areas of the ocean are accumulating high levels of waste and pollutants. We now understand that any kind of pollution eventually ends up in the ocean. It may be carried by the wind, freshwater rivers

and/or streams, coastal erosion, or watercraft of any sort. We have even found the most remote places on earth to be impacted. However, the true extent and the impacts of our wastes are poorly understood. One reason for this lack of understanding is due to the difficulties the ocean environment poses. Many places are geographically difficult to access; these areas require a large financial, technical, personnel and personal burden to study. In the rare cases that these obstacles are overcome, there remain the additional challenges of contending with environmental challenges (e.g. weather) and the simple fact that marine life is difficult to see, and can only be observed for a short period of time. Finally, the vast majority of marine animals cannot be used in laboratory experiments, and we do not currently have a useful model species to use in their place. Yet, despite these difficulties there are ways to study the impacts of pollution on marine life without sacrificing a large number of organisms and threatening their populations.

Skin Biopsy Applications in Free Ranging Marine Mammals: A Case Study of Whale Skin Biopsies

Skin biopsies can provide a wealth of information about marine mammal pollution. Here we have shown how chromium, a little studied chemical in the marine environment, has contaminated whales in even the remotest regions. Using the levels of chromium we reported in the biopsies we have determined that the treatment concentrations that induced DNA damage in our cell culture toxicological analysis were orders of magnitude lower. To add further supporting context, human workers exposed occupationally to Cr(VI) had levels of chromium in lung tissue of 20.4 ug/g [9] while levels of skin were reported to be 0.05 ug/g [10]. These workers died of lung cancer. Considering that the average skin levels found in sperm whales and North Atlantic right whales of 9.3 ug/g and 7 ug/g, these levels appear to

be extremely high compared to occupationally exposed humans and are of concern.

Catherine F. Wise, John Pierce Wise, Jr., Sandra S. Wise and John Pierce Wise, Sr *Wise Laboratory of Environmental and Genetic Toxicology, Maine Center for Toxicology and* 

We would like to thank Tania Li Chen, James Wise, Christy Gianios Jr., Hong Xie, Amie Holmes, Carolyne LaCerte, Shouping Huang, Julieta Martino, Fariba Shaffiey, Kaitlynn M. Levine, Marijke Grau, W. Douglas Thompson, Christopher Perkins, AbouEl-Makarim Aboueissa, Tongzhang Zheng, Scott Kraus, Yawei Zhang, Tracy Romano, Todd O'Hara, Cairong Zhu, Lucille Benedict, Gregory Buzard, Iain Kerr, Bob Wallace, Jeffrey Kunz, John Atkinson, Roger Payne and all of the Odyssey boat crew for technical assistance and

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Environmental Health Sciences (NIEHS), the National Institutes of Health, the Army Research Office, the Department of Defense, the Environmental Protection Agency, the United States Department of Commerce or the National Oceanic and Atmospheric Administration (NOAA). It has not been formally reviewed by EPA or any other federal agency. The EPA and other federal agencies do not endorse any products or commercial services mentioned in this chapter. Work was conducted under NMFS permit #1008-1637-03 (J. Wise Sr., PI) and permit #751-1614 (Iain

*Environmental Health, University of Southern Maine, Portland Maine, USA* 

oceans.

**Author details** 

**Acknowledgement** 

professional advice.

By measuring levels of metals and other pollutants we can inform our in vitro toxicological experiments by using more environmentally relevant treatment concentrations. In addition, we have used the biopsies to develop cell lines to test potential environmental pollutants like chromium in a species specific model rather than use human or mouse models to inform on pollutants specifically being exposed to a marine mammal population. These results demonstrate that environmental pollutants found in the ocean are accumulating in the whales and are likely to have important health repercussions to the animals living in the

as a Valuable and Essential Tool for Studying Marine Mammal Toxicology and Conservation 121

Thus, marine mammals provide the best model for studying marine pollution. They are found near-shore (e.g. sea otters, seals, sea lions, sirenians, and some cetaceans) and offshore (e.g. most cetaceans, some seals, and some sea lions), and their diets range the entirety of the food chain from plankton and krill to giant squid and other marine mammals. A handful of marine mammals have cosmopolitan or near-cosmopolitan distribution, which enables for a more controlled comparison of marine pollution in different areas; these include sperm whales, bottlenose dolphins, orcas, and humpback whales. All marine mammals attract ecotourism, thus there are many watercraft that seek them out for observation. Along with these watercraft come the exhaust from burning fossil fuels, noise pollution that can interfere with the animals' communication and any trash that may be accidently lost overboard.

The health of all marine mammals depends on clean air and water; they all live in the water exclusively or near-exclusively, and since they are mammals they need air to breathe. Not all are directly affected by pollution in the sediment, but may still be indirectly affected through their prey. Marine mammal prey is highly varied from species to species. Studying a variety of them, we can determine if the effect of pollution is specific to animals that are higher on the food chain (likely caused by biomagnification), or if the effect is conserved throughout the food chain (likely caused by bioaccumulation).

Furthermore, marine mammals are suitable organisms to study because they capture the attention and support of the mass media and general public. These animals are one of the major bases for marine ecotourism because of their charismatic behaviors and their similarities with humans; they breathe air, are warm-blooded, nurse their young, and communicate with songs and chatter. Their similarities also enable them to be studied as a model species for humans [8]. The ocean is a finite resource, and has a finite capacity for pollution intake. Marine mammals can serve as a model species for humans to demonstrate how we will be affected by marine pollution in the future if it is not monitored and regulated better. They spend either all or nearly all their lives in the ocean, and they will exhibit impacts by marine pollution sooner than humans will.

Skin biopsies can provide a wealth of information about marine mammal pollution. Here we have shown how chromium, a little studied chemical in the marine environment, has contaminated whales in even the remotest regions. Using the levels of chromium we reported in the biopsies we have determined that the treatment concentrations that induced DNA damage in our cell culture toxicological analysis were orders of magnitude lower. To add further supporting context, human workers exposed occupationally to Cr(VI) had levels of chromium in lung tissue of 20.4 ug/g [9] while levels of skin were reported to be 0.05 ug/g [10]. These workers died of lung cancer. Considering that the average skin levels found in sperm whales and North Atlantic right whales of 9.3 ug/g and 7 ug/g, these levels appear to be extremely high compared to occupationally exposed humans and are of concern.

By measuring levels of metals and other pollutants we can inform our in vitro toxicological experiments by using more environmentally relevant treatment concentrations. In addition, we have used the biopsies to develop cell lines to test potential environmental pollutants like chromium in a species specific model rather than use human or mouse models to inform on pollutants specifically being exposed to a marine mammal population. These results demonstrate that environmental pollutants found in the ocean are accumulating in the whales and are likely to have important health repercussions to the animals living in the oceans.
